Project Summary The ultimate goal of this project is developing a new class of broad spectrum antibiotics, focused on Gram- negative bacteria. Inspired by a natural product, this class targets an unexploited binding site of the bacterial ribosome that selectively inhibits bacterial protein synthesis. With ever increasing reports of resistance to frontline therapies addressing Gram-negatives, there is a critical need for new therapies, yet very little can be found in the development pipeline. As a result, any new therapeutic that targets Gram-negative bacteria will address an unmet medical need. Crystal structures of the natural product and C?rza analogs bound to the T. thermophilus ribosome have been used to guide medicinal chemistry efforts. These crystal structures revealed that a highly conserved region of the peptidyl transferase center (PTC) that has yet to be exploited by any existing antibiotics is in fact a viable new therapeutic approach against a well-vetted target, the ribosome. Binding to this site of the PTC appears to convey bacterial selectivity. Molecular modeling using these complexes as a guide for preliminary optimization has produced new leads that have expanded the antibacterial spectrum of activity from solely Mtb to include potent activity against E. coli, K. pneumoniae and S. aureus (including drug-resistant strains for each of these). These analogs inhibit protein synthesis in the nM concentration range, exhibit selectivity indexes of >400 for inhibition of bacterial versus eukaryotic protein synthesis and have very limited toxicity to mammalian cells. This five-year R01 project will ultimately develop a new potent, broad spectrum antibacterial drug candidate, focusing on Gram-negative pathogens ready for IND preparation. Development of this antibacterial lead series will be accomplished by the following aims. Aim 1 will optimize the lead series to expand activity to additional Gram-negative pathogens, notably P. aeruginosa and A. baumannii. Aim 2 will define mechanism of action, biochemical/microbiological activity and in vitro ADME-Tox of analogs by evaluating activity against bacterial and eukaryotic protein synthesis (including mitochondrial), a panel of bacteria representing Gram-negative and Gram-positive organisms (including antibiotic-resistant). Preliminary ADME-Tox will be evaluated in vitro using Vero cells to predict cytotoxicity, microsomal stability, inhibition of hERG and cytochrome P450s, Ames test for genotoxicity and in an in vitro receptor binding assay of 44 primary molecular targets. Aim 3 will use in vivo PK to guide optimization followed by determining the MTD and in vivo efficacy in a mouse thigh infection model to select a single lead compound for advancement. Aim 4 will expand the microbiological profile by determining MIC90s, characterization of resistance and potential cross-resistance to other antibiotics. Aim 5 will expand in vivo efficacy and PK/PD to pneumonia infection models. Aim 6 will address CMC issues and produce an initial cGMP lot for use in GLP toxicology studies in Aim 7.